LETTER TO THE EDITOR

Predictable difficulty or difficulty to predict

Predicting the functional consequence of a given

amino acid replacement, that is, the difference

between the wild type protein and its nonsynony-

mous single nucleotide polymorph variants (nsSNPs)

is a great challenge. This is especially true in the

case of large membrane proteins, like ABC trans-

porters. Kelly et al. has published such an ambitious

study in a recent issue of Protein Science focusing

on nsSNP in structurally conserved segments within

the nucleotide binding domains (NBDs) of ABC

transporters supposed to be involved in interdomain

communication.1 With the aid of structural rationali-

zation the authors predicted the impact of 40

nsSNPs found in seven clinically important human

ABC transporters using a bivalent scoring: disease

or neutral.

We have performed a deep search of the avail-

able literature to find published data on the 40

nsSNPs to delineate them with the predictions. We

have incorporated only data from original peer-

reviewed publications into our dataset, and our hits

were checked in various gene-specific databases too

[BioBase and ABCC6 Database]. Based on the avail-

able information, we categorized the published data

on a given nsSNP as (i) ‘‘disease-associated’’ when

the publication clearly demonstrates that the vari-

ant segregates with the disease but is absent from

at least 200 alleles of unrelated, nonaffected individ-

uals; or (ii) in vitro experimental evidence when the

specific variant was heterologously expressed and its

(transport) function and/or folding-stability was

investigated (Table I). We considered functional dif-

ferences only if the level of expression of the given

nsSNP variant was comparable to the wild type

expressed in the same system. Our results, in com-

parison with the predictions of Kelly et al. are sum-

marized in Table I. The left five columns of the table

is taken from the publication of Kelly et al. On the

right we list our findings and give the related pri-

mary reference. In several cases more than one inde-

pendent studies were found showing the same

result. To keep the list of references short, we indi-

cated only one reference, if more publications were

found we added þn (where n is the number of addi-

tional independent publications).

We found the published data on 19 of the 40

ABC nsSNPs. Of these, 16 nsSNPs had been tested

in in vitro experiments and in seven examples the

given nsSNP resulted in impaired function and/or

folding. In one case the published nsSNP generates

a new splice site (exon skipping), what affects the

mRNA maturation therefore the function or folding

of the protein with the given nsSNP can not be con-

cluded (ABCB11, E1186K). One of the nsSNPs was

characterized solely by its association to a genetic

disease (ABCC6, A1291T associated to pseudoxan-

thoma elasticum). One additional nsSNP was found

as homozygous nsSNP in several healthy individuals

therefore it was categorized as neutral, on the basis

of this human genetic data (ABCC6, R1268Q). There

are two nsSNPs with both published genetic associa-

tion and in vitro experimental characterization in

our data set (ABCB11 V444A associated to intrahe-

patic cholestasis of pregnancy; ABCG2, Q141K asso-

ciated to gout).

Our conclusions on the nsSNPs are listed in col-

umn ‘‘Published phenotype’’ of Table I.; for the sake

of simplicity we kept the bivalent terminology of

Kelly et al.: disease or neutral. Comparing the pre-

dicted phenotype of the Kelly’s paper with the phe-

notype extracted from the published data reveals

misprediction in roughly half of the cases (10/19)

thus demonstrating the inherent difficulties of

rationalizing and predicting the functional impact of

snSNPs.

Acknowledgment

This work was supported by the Hungarian research

grants OTKA CK80135, OTKA NK81204, OTKA

PD79783 and by NIH R01AR055225 (subaward) to

A.V. A.T. is a recipient of Bolyai Fellowship of the Hun-

garian Academy of Sciences.

TAMAS ARANYI

KRISZTINA FULOP

ORSOLYA SYMMONS

VIOLA POMOZI

ANDRAS VARADI*Institute of EnzymologyHungarian Academy of SciencesBudapest, Hungary(Received 20 October 2010)

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*Correspondence to: Andras Varadi, E-mail: varadi@enzim.hu

DOI: 10.1002/pro.552

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Table I. Predicted and Published Phenotype of nsSNPs in Human ABC Transporters

Commonname

HUGOname Mutation NBD Predicted1

Publishedphenotype Disease-asscociated

Protein studiedin vitro Reference

BSEP ABCB11 E592Q NBD1 NEUTRAL Not available — — —BSEP ABCB11 N591S NBD1 NEUTRAL NEUTRAL — Not affected 2 þ 3BSEP ABCB11 Q558H NBD1 NEUTRAL Not available — — —BSEP ABCB11 V444A NBD1 NEUTRAL DISEASE Assoc. to ICP Impaired 3BSEP ABCB11 E1186K NBD2 DISEASE Not applicable Exon skipping Not applicable 3 þ 2MDR1 ABCB1 P1051A NBD2 NEUTRAL NEUTRAL — Not affected 4MDR1 ABCB1 S1141T NBD2 NEUTRAL DISEASE — Gain of function 5MDR1 ABCB1 T1256K NBD2 DISEASE Not available — — —MDR1 ABCB1 V1251I NBD2 NEUTRAL DISEASE — Gain of function 6MDR1 ABCB1 W1108R NBD2 DISEASE DISEASE — Impaired 5MRP2 ABCC2 I670T NBD1 DISEASE Not available — — —MRP2 ABCC2 L849R NBD1 DISEASE Not available — — —MRP2 ABCC2 C1515Y NBD2 DISEASE Not available Not assoc. to ICP — 7MRP3 ABCC3 D770N NBD1 NEUTRAL Not available — — —MRP3 ABCC3 K718M NBD1 NEUTRAL Not available — — —MRP3 ABCC3 T809M NBD1 DISEASE Not available — — —MRP3 ABCC3 V765L NBD1 DISEASE NEUTRAL — Not affected 8MRP3 ABCC3 Q1365R NBD2 DISEASE NEUTRAL — Not affected 8MRP3 ABCC2 R1297H NBD2 DISEASE Not available — — —MRP3 ABCC3 R1348C NBD2 DISEASE NEUTRAL — Not affected 8MRP3 ABCC3 R1381S NBD2 DISEASE DISEASE — Impaired 8MRP4 ABCC4 G487E NBD1 DISEASE DISEASE — Impaired 9MRP4 ABCC4 K498E NBD1 NEUTRAL NEUTRAL — Not affect. 9MRP4 ABCC4 R1220Q NBD2 NEUTRAL Not available — — —MRP4 ABCC4 T1142M NBD2 NEUTRAL Not available — — —MRP4 ABCC4 V1071I NBD2 NEUTRAL NEUTRAL — Not affected 9MRP6 ABCC6 I1330L NBD2 NEUTRAL Not available — — —MRP6 ABCC6 I742V NBD1 NEUTRAL Not available — —MRP6 ABCC6 P664S NBD1 NEUTRAL Not available — — —MRP6 ABCC6 R724K NBD1 NEUTRAL NEUTRAL Not assoc. to PXE — 10MRP6 ABCC6 R769K NBD1 NEUTRAL Not available — — —MRP6 ABCC6 A1291T NBD2 NEUTRAL DISEASE Assoc. to PXE — 11MRP6 ABCC6 E1369K NBD2 NEUTRAL Not available — — —MRP6 ABCC6 G1327E NBD2 DISEASE Not available — — —MRP6 ABCC6 L1416R NBD2 DISEASE Not available — — —MRP6 ABCC6 R1268Q NBD2 DISEASE NEUTRAL Homozygous

not assoc. to PXE— 12

MRP6 ABCC6 R1461H NBD2 DISEASE Not available — — —MXR ABCG2 I206L NBD1 NEUTRAL NEUTRAL — Not affected 13 þ 2MXR ABCG2 P269S NBD1 DISEASE NEUTRAL — Not affected 14MXR ABCG2 Q141K NBD1 NEUTRAL DISEASE Yes/gout Impaired 15 þ 5

The left panel of the table (five column) is taken from Ref. 1. On the right we list our findings and their related primaryreference. In several cases, we found more than one independent study showing the same result. To keep the list of referen-ces short, we indicated only one reference, if more publications were found we added þn (where n is the number of addi-tional independent publications). Neutral and disease phenotypes are shown in blue and red, respectively.ICP, intrahepatic cholestasis of pregnancy; PXE, pseudoxanthoma elasticum.

2 PROTEINSCIENCE.ORG Letter to the Editor

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